Eukaryotic organisms, including animals and plants, have evolved sophisticated immune systems to protect themselves against pathogens. Small RNAs (sRNAs) are short, non-coding regulatory RNAs that induce RNA interference (RNAi) by binding to Argonaute (AGO) proteins and directing silencing of genes with complementary sequences. Our work and those of others have demonstrated that sRNAs, derived from either hosts or pathogens, function through AGO proteins and play an important role in host-pathogen interactions. However, the underlying regulatory mechanisms remain elusive. Our long-term goal is to understand the dynamic regulation of sRNAs and RNAi in host innate immunity. AGO proteins are key players of sRNA- mediated gene silencing, and we hypothesize that they are modified and regulated by their associated proteins to modulate sRNA-mediated gene silencing. Here, we seek to understand the modification and regulation of AGOs in RNAi pathway, and in host antibacterial and antifungal immune responses, by using model plant-pathogen interaction systems - Arabidopsis thaliana and its bacterial pathogen Pseudomonas syringae and aggressive fungal pathogen Botrytis cinerea. During the last funding period, we have identified diverse classes of host sRNAs regulating host immunity, and have demonstrated that Arabidopsis AGO2 (AtAGO2) is highly induced by bacterial infection and positively regulates host immunity. We have also shown that B. cinerea delivers its sRNAs into host cells and hijacks host AtAGO1 to suppress host immunity genes for successful infection. To obtain a mechanistic understanding of how AtAGO1 and AtAGO2 are regulated, we have identified their associated proteins via proteomics analysis. This renewal will study the modification and regulation of AtAGO1 and AtAGO2 by their associated proteins in RNAi pathways and host innate immunity. The output of this study will significantly advance our understanding of dynamic control of RNAi in host immune responses against pathogens, which will have great impact on both agricultural and human health. The specific aims are: 1. Elucidate the role of protein arginine methyltransferases in modification and regulation of AtAGO function in host immunity. 2. Investigate the function of DDX3-like RNA helicases in modulating AtAGO function in host immunity. 3. Study the role of protein phosphatases in modulating AtAGO and host immunity.